Numerous TH-positive bouton-rich fibers traversed the STN. Immunohistochemistry revealed that most of the subthalamic neurons were glutamatergic and differed significantly in appearance from the large stellate TH-positive cells of the adjacent SNc. The neurons in the STN had medium-sized (25–40 μm) ovoid or fusiform cell bodies, from which three to six large dendrites emanated in a direction predominantly parallel to the long axis of the STN. Rostrally, the STN approached the globus pallidus pars interna, whereas caudally the ventromedial part of the STN was adjacent to the rostral part of the substantia nigra pars compacta (SNc), where some of the neurons of the two nuclei merged. In all of the stained preparations the STN appeared as a distinct lens-shaped structure located in the caudal diencephalon, medial to the internal capsule and ventrolateral to the zona incerta. The organization of several neurotransmitters in the STN has been evaluated in histological preparations stained for acetylcholinesterase (AChE) and immunostained for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), glutamic acid decarboxylase (GAD) and glutamate. It is based on histological material stained with Nissl, Golgi and autometallographic techniques, and employs design-based stereological estimation of the total neuron number. This study provides a light-microscopic description of the organization, morphology and number of neurons in the subthalamic nucleus (STN) of the Göttingen minipig.